Primer sequences for RT-qPCR reaction.

Osteoporosis is an important health problem that occurs due to an imbalance between bone formation and resorption. Hormonal deficiency post-menopause is a significant risk factor. The probiotic Limosilactobacillus reuteri has been reported to prevent ovariectomy (Ovx)-induced bone loss in mice and reduce bone loss in postmenopausal women. Despite the numerous health benefits of probiotics, as they are live bacteria, the administration is not risk-free for certain groups (e.g., neonates and immunosuppressed patients). We evaluated the effects of L. reuteri (ATCC PTA 6475) and its heat-killed (postbiotic) form on Ovx-induced bone loss. Adult female mice (BALB/c) were randomly divided into four groups: group C—control (sham); group OVX-C—Ovx; group OVX-POS—Ovx + heat-killed probiotic; group OVX-PRO—Ovx + probiotic. L. reuteri or the postbiotic was administered to the groups (1.3x109 CFU/day) by gavage. Bacterial morphology after heat treatment was accessed by scanning electron microscopy (SEM). The treatment started one week after Ovx and lasted 28 days (4 weeks). The animals were euthanized at the end of the treatment period. Bone microarchitecture and ileum Occludin and pro-inflammatory cytokines gene expression were evaluated by computed microtomography and qPCR techniques, respectively. The Ovx groups had lower percentage of bone volume (BV/TV) and number of bone trabeculae as well as greater total porosity compared to the control group. Treatment with live and heat-killed L. reuteri resulted in higher BV/TV and trabecular thickness than the Ovx group. The heat treatment caused some cell surface disruptions, but its structure resembled that of the live probiotic in SEM analysis. There were no statistical differences in Occludin, Il-6 and Tnf-α gene expression. Both viable and heat-killed L. reuteri prevented bone loss on ovariectomized mice, independently of gut Occludin and intestinal Il-6 and Tnf-α gene expression.

骨质疏松症（Osteoporosis）是一类因骨形成与骨吸收失衡引发的重要健康问题。绝经后激素缺乏是其显著危险因素。已有研究证实，益生菌罗伊氏乳杆菌（Limosilactobacillus reuteri）可预防卵巢切除术（ovariectomy, Ovx）诱导的小鼠骨丢失，并减轻绝经后女性的骨量丢失。尽管益生菌具备诸多健康益处，但由于其为活菌体，对新生儿、免疫功能低下患者等特定人群而言，服用益生菌并非无风险。本研究评估了罗伊氏乳杆菌（L. reuteri, ATCC PTA 6475）及其热灭活形式（后生元，postbiotic）对Ovx诱导骨丢失的影响。将成年雌性BALB/c小鼠随机分为4组：C组为假手术对照组；OVX-C组为卵巢切除术模型组；OVX-POS组为卵巢切除术+热灭活益生菌组；OVX-PRO组为卵巢切除术+活益生菌组。通过灌胃方式对各组小鼠给予1.3×10^9 CFU/天的罗伊氏乳杆菌或后生元。采用扫描电子显微镜（scanning electron microscopy, SEM）观察热处理后菌体的形态结构。干预于卵巢造模术后1周启动，持续28天（共4周）。干预结束后对所有小鼠实施安乐死。分别采用显微计算机断层扫描（computed microtomography）与实时荧光定量聚合酶链式反应（qPCR）技术，检测小鼠骨微结构以及回肠紧密连接蛋白Occludin与促炎细胞因子的基因表达水平。与假手术对照组相比，卵巢切除术模型组的骨体积分数（bone volume/total volume, BV/TV）、骨小梁数量均更低，总孔隙度更高。活菌体与热灭活罗伊氏乳杆菌干预后，小鼠的BV/TV及骨小梁厚度均较卵巢切除术模型组显著升高。扫描电镜观察显示，热处理虽造成菌体表面出现一定程度的结构破坏，但整体形态与活益生菌仍较为相似。各组小鼠回肠Occludin、IL-6及TNF-α的基因表达水平均无统计学差异。无论活菌形式还是热灭活形式的罗伊氏乳杆菌，均可预防卵巢切除小鼠的骨量丢失，且该作用与肠道Occludin及肠内IL-6、TNF-α的基因表达无关。